Potential of the Osteoclast’s Proton Pump as a Drug Target in Osteoporosis

Decreasing bone mass during aging predisposes to fractures and it is estimated that every second woman and one in five men will suffer osteoporotic fractures during their lifetime. Bone is an adaptive tissue undergoing continuous remodeling in response to physical and metabolic stimuli. Bone mass decreases through a net negative balance in the bone remodeling process of bone, in which the new bone incompletely replaces the resorbed bone mass. Bone resorption is carried out by the osteoclasts; the bone mineral is solubilized by acidification and the organic matrix is subsequently degraded by proteases. Several classes of drugs are available for prevention of osteoporotic fractures. They act by different mechanisms to increase bone mass, and some of them act mainly as antiresorptives by inhibition of osteoclast formation or their function. Optimally, a drug should act selectively on a specific process, since other processes affected usually result in adverse effects. The purpose of this study was to evaluate whether the osteoclastic vacuolar adenosine trisphosphatases (V-ATPase), which drives the solubilization of bone mineral, can be selectively inhibited despite its ubiquitous cellular functions. The V-ATPase is a multimeric protein composed of 13 subunits of which six possesses two or more isoforms. Selectivity for the osteoclastic V-ATPase could be provided if it has some structural uniqueness, such as a unique isoform combination. The a3 isoform of the 116kDa subunit is inevitable for bone resorption; however, it is also present in, and mainly limited to, the lysosomes of other cells. No evidence of a structural uniqueness of the osteoclastic V-ATPase compared to the lysosomal V-ATPase was found, although this can not yet be excluded. Thus, an inhibitor selective for the a3 isoform would target the lysosomal V-ATPase as well. However, the results suggest that selectivity for bone resorption over lysosomal function can be obtained by two other mechanisms, suggesting that isoform a3 is a valid target. The first is differential compensation; bone resorption depends on the high level of a3 expression, and is not compensated for by other isoforms, while the lower level of a3 in lysosomes of other cells may be partly compensated for. The second mechanism is because the bone resorption process itself is fundamentally different from lysosomal acidification because of the chemistry of bone dissolution and the anatomy of the resorbing osteoclast. By this mechanism, full inhibition of bone resorption is obtained with more than tenfold lower inhibitor concentration than those needed to fully inhibit lysosomal acidification. The two mechanisms are additive. Based on the results, we suggest that bone resorption can be selectively inhibited if VATPase inhibitors that are sufficiently selective for the a3 isoform over the other isoforms are developed.

A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

VAP-1 as drug target in inflammation : structural features determining ligand interactions

Vascular adhesion protein-1 (VAP-1), which belongs to the copper amine oxidases (CAOs), is a validated drug target in inflammatory diseases. Inhibition of VAP-1 blocks the leukocyte trafficking to sites of inflammation and alleviates inflammatory reactions. In this study, a novel set of potent pyridazinone inhibitors is presented together with their X-ray structure complexes with VAP-1. The crystal structure of serum VAP-1 (sVAP-1) revealed an imidazole binding site in the active site channel and, analogously, the pyridazinone inhibitors were designed to bind into the channel. This is the first time human VAP-1 has been crystallized with a reversible inhibitor and the structures reveal detailed information of the binding mode on the atomic level. Similarly to some earlier studied inhibitors of human VAP-1, the designed pyridazinone inhibitors bind rodent VAP-1 with a lower affinity than human VAP-1. Therefore, we made homology models of rodent VAP-1 and compared human and rodent enzymes to determine differences that might affect the inhibitor binding. The comparison of the crystal structures of the human VAP-1 and the mouse VAP-1 homology model revealed key differences important for the species specific binding properties. In general, the channel in mouse VAP-1 is more narrow and polar than the channel in human VAP-1, which is wider and more hydrophobic. The differences are located in the channel leading to the active site, as well as, in the entrance to the active site channel. The information obtained from these studies is of great importance for the development and design of drugs blocking the activity of human VAP-1, as rodents are often used for in vivo testing of candidate drugs. In order to gain more insight into the selective binding properties of the different CAOs in one species a comprehensive evolutionary study of mammalian CAOs was performed. We found that CAOs can be classified into sub-families according to the residues X1 and X2 of the Thr/Ser-X1-X2-Asn-Tyr-Asp active site motif. In the phylogenetic tree, CAOs group into diamine oxidase, retina specific amine oxidase and VAP-1/serum amine oxidase clades based on the residue in the position X2. We also found that VAP-1 and SAO can be further differentiated based on the residue in the position X1. This is the first large-scale comparison of CAO sequences, which explains some of the reasons for the unique substrate specificities within the CAO family.

Role of human hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) in steroid-dependent diseases in females –novel indications for HSD17B1 inhibitors. Phenotypic analysis of transgenic mice overexpressing human HSD17B1.

Hormone-dependent diseases, e.g. cancers, rank high in mortality in the modern world, and thus, there is an urgent need for new drugs to treat these diseases. Although the diseases are clearly hormone-dependent, changes in circulating hormone concentrations do not explain all the pathological processes observed in the diseased tissues. A more inclusive explanation is provided by intracrinology – a regulation of hormone concentrations at the target tissue level. This is mediated by the expression of a pattern of steroid-activating and -inactivating enzymes in steroid target tissues, thus enabling a concentration gradient between the blood circulation and the tissue. Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) form a family of enzymes that catalyze the conversion between low active 17-ketosteroids and highly active 17beta-hydroxysteroids. HSD17B1 converts low active estrogen (E1) to highly active estradiol (E2) with high catalytic efficiency, and altered HSD17B1 expression has been associated with several hormone-dependent diseases, including breast cancer, endometriosis, endometrial hyperplasia and cancer, and ovarian epithelial cancer. Because of its putative role in E2 biosynthesis in ovaries and peripheral target tissues, HSD17B1 is considered to be a promising drug target for estrogen-dependent diseases. A few studies have indicated that the enzyme also has androgenic activity, but they have been ignored. In the present study, transgenic mice overexpressing human HSD17B1 (HSD17B1TG mice) were used to study the effects of the enzyme in vivo. Firstly, the substrate specificity of human HSD17B1 was determined in vivo. The results indicated that human HSD17B1 has significant androgenic activity in female mice in vivo, which resulted in increased fetal testosterone concentration and female disorder of sexual development appearing as masculinized phenotype (increased anogenital distance, lack of nipples, lack of vaginal opening, combination of vagina with urethra, enlarged Wolffian duct remnants in the mesovarium and enlarged female prostate). Fetal androgen exposure has been linked to polycystic ovary syndrome (PCOS) and metabolic syndrome during adulthood in experimental animals and humans, but the genes involved in PCOS are largely unknown. A putative mechanism to accumulate androgens during fetal life by HSD17B1 overexpression was shown in the present study. Furthermore, as a result of prenatal androgen exposure locally in the ovaries, HSD17B1TG females developed ovarian benign serous cystadenomas in adulthood. These benign lesions are precursors of low-grade ovarian serous tumors. Ovarian cancer ranks fifth in mortality of all female cancers in Finland, and most of the ovarian cancers arise from the surface epithelium. The formation of the lesions was prevented by prenatal antiandrogen treatment and by transplanting wild type (WT) ovaries prepubertally into HSD17B1TG females. The results obtained in our non-clinical TG mouse model, together with a literature analysis, suggest that HSD17B1 has a role in ovarian epithelial carcinogenesis, and especially in the development of serous tumors. The role of androgens in ovarian carcinogenesis is considered controversial, but the present study provides further evidence for the androgen hypothesis. Moreover, it directly links HSD17B1-induced prenatal androgen exposure to ovarian epithelial carcinogenesis in mice. As expected, significant estrogenic activity was also detected for human HSD17B1. HSD17B1TG mice had enhanced peripheral conversion of E1 to E2 in a variety of target tissues, including the uterus. Furthermore, this activity was significantly decreased by treatments with specific HSD17B1 inhibitors. As a result, several estrogen-dependent disorders were found in HSD17B1TG females. Here we report that HSD17B1TG mice invariably developed endometrial hyperplasia and failed to ovulate in adulthood. As in humans, endometrial hyperplasia in HSD17B1TG females was reversible upon ovulation induction, triggering a rise in circulating progesterone levels, and in response to exogenous progestins. Remarkably, treatment with a HSD17B1 inhibitor failed to restore ovulation, yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment. We also demonstrate that HSD17B1 is expressed in normal human endometrium, hyperplasia, and cancer. Collectively, our non-clinical data and literature analysis suggest that HSD17B1 inhibition could be one of several possible approaches to decrease endometrial estrogen production in endometrial hyperplasia and cancer. HSD17B1 expression has been found in bones of humans and rats. The non-clinical data in the present study suggest that human HSD17B1 is likely to have an important role in the regulation of bone formation, strength and length during reproductive years in female mice. Bone density in HSD17B1TG females was highly increased in femurs, but in lesser amounts also in tibias. Especially the tibia growth plate, but not other regions of bone, was susceptible to respond to HSD17B1 inhibition by increasing bone length, whereas the inhibitors did not affect bone density. Therefore, HSD17B1 inhibitors could be safer than aromatase inhibitors in regard to bone in the treatment of breast cancer and endometriosis. Furthermore, diseases related to improper growth, are a promising new indication for HSD17B1 inhibitors.

Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapy

ERBB4 mutations in cancer and amyotrophic lateral sclerosis

ErbB receptor tyrosine kinases, epidermal growth factor receptor (EGFR, also known as ErbB1), ErbB2 (HER2 or NEU), ErbB3 (HER3), and ErbB4 (HER4), transduce signals borne by extracellular ligands into central cellular responses such as proliferation, survival, differentiation, and apoptosis. Mutations in ERBB genes are frequently detected in human malignant diseases of epithelial and neural origin, making ErbB receptors important drug targets. Targeting EGFR and ErbB2 has been successful in eg. lung and breast cancer, respectively, and mutations in these genes can be used to select patients that are responsive to the targeted treatment. Although somatic ERBB4 mutations have been found in many high-incidence cancers such as melanoma, lung cancer, and colorectal cancer and germ-line ERBB4 mutations have been linked to neuronal disorders and cancer, ErbB4 has generally been neglected as a potential drug target. Thus, the consequences of ERBB4 mutations on ErbB4 biology are largely unknown. This thesis aimed to elucidate the functional consequences and assess the clinical significance of somatic and germ-line ERBB4 mutations in the context of cancer and amyotrophic lateral sclerosis. The results of this study indicated that cancer-associated ERBB4 mutations can promote aberrant ErbB4 function by activating the receptor or inducing qualitative changes in ErbB4 signaling. ERBB4 mutations increased survival or decreased differentiation in vitro, suggesting that ERBB4 mutations can be oncogenic. Importantly, the potentially oncogenic mutations were located in various subdomains in ErbB4, possibly providing explanation for the characteristic scattered pattern of mutations in ERBB4. This study also demonstrated that hereditary variation in ERBB4 gene can have a significant effect on the prognosis of breast cancer. In addition, it was shown that hereditary or de novo germ-line ERBB4 mutations that predispose to amyotrophic lateral sclerosis inhibit ErbB4 activity. Together, these results suggest that ErbB4 should be considered as a novel drug target in cancer and amyotrophic lateral sclerosis.

Cell Model Systems in Characterizing Alpha2-Adrenoceptors as Drug Targets.

The three alpha2-adrenoceptor (alpha2-AR) subtypes belong to the G protein-coupled receptor superfamily and represent potential drug targets. These receptors have many vital physiological functions, but their actions are complex and often oppose each other. Current research is therefore driven towards discovering drugs that selectively interact with a specific subtype. Cell model systems can be used to evaluate a chemical compound's activity in complex biological systems. The aim of this thesis was to optimize and validate cell-based model systems and assays to investigate alpha2-ARs as drug targets. The use of immortalized cell lines as model systems is firmly established but poses several problems, since the protein of interest is expressed in a foreign environment, and thus essential components of receptor regulation or signaling cascades might be missing. Careful cell model validation is thus required; this was exemplified by three different approaches. In cells heterologously expressing alpha2A-ARs, it was noted that the transfection technique affected the test outcome; false negative adenylyl cyclase test results were produced unless a cell population expressing receptors in a homogenous fashion was used. Recombinant alpha2C-ARs in non-neuronal cells were retained inside the cells, and not expressed in the cell membrane, complicating investigation of this receptor subtype. Receptor expression enhancing proteins (REEPs) were found to be neuronalspecific adapter proteins that regulate the processing of the alpha2C-AR, resulting in an increased level of total receptor expression. Current trends call for the use of primary cells endogenously expressing the receptor of interest; therefore, primary human vascular smooth muscle cells (SMC) expressing alpha2-ARs were tested in a functional assay monitoring contractility with a myosin light chain phosphorylation assay. However, these cells were not compatible with this assay due to the loss of differentiation. A rat aortic SMC cell line transfected to express the human alpha2B-AR was adapted for the assay, and it was found that the alpha2-AR agonist, dexmedetomidine, evoked myosin light chain phosphorylation in this model.

Influence of surface functionalization on the behavior of silica nanoparticles in biological systems

Personalized nanomedicine has been shown to provide advantages over traditional clinical imaging, diagnosis, and conventional medical treatment. Using nanoparticles can enhance and clarify the clinical targeting and imaging, and lead them exactly to the place in the body that is the goal of treatment. At the same time, one can reduce the side effects that usually occur in the parts of the body that are not targets for treatment. Nanoparticles are of a size that can penetrate into cells. Their surface functionalization offers a way to increase their sensitivity when detecting target molecules. In addition, it increases the potential for flexibility in particle design, their therapeutic function, and variation possibilities in diagnostics. Mesoporous nanoparticles of amorphous silica have attractive physical and chemical characteristics such as particle morphology, controllable pore size, and high surface area and pore volume. Additionally, the surface functionalization of silica nanoparticles is relatively straightforward, which enables optimization of the interaction between the particles and the biological system. The main goal of this study was to prepare traceable and targetable silica nanoparticles for medical applications with a special focus on particle dispersion stability, biocompatibility, and targeting capabilities. Nanoparticle properties are highly particle-size dependent and a good dispersion stability is a prerequisite for active therapeutic and diagnostic agents. In the study it was shown that traceable streptavidin-conjugated silica nanoparticles which exhibit a good dispersibility could be obtained by the suitable choice of a proper surface functionalization route. Theranostic nanoparticles should exhibit sufficient hydrolytic stability to effectively carry the medicine to the target cells after which they should disintegrate and dissolve. Furthermore, the surface groups should stay at the particle surface until the particle has been internalized by the cell in order to optimize cell specificity. Model particles with fluorescently-labeled regions were tested in vitro using light microscopy and image processing technology, which allowed a detailed study of the disintegration and dissolution process. The study showed that nanoparticles degrade more slowly outside, as compared to inside the cell. The main advantage of theranostic agents is their successful targeting in vitro and in vivo. Non-porous nanoparticles using monoclonal antibodies as guiding ligands were tested in vitro in order to follow their targeting ability and internalization. In addition to the targeting that was found successful, a specific internalization route for the particles could be detected. In the last part of the study, the objective was to clarify the feasibility of traceable mesoporous silica nanoparticles, loaded with a hydrophobic cancer drug, being applied for targeted drug delivery in vitro and in vivo. Particles were provided with a small molecular targeting ligand. In the study a significantly higher therapeutic effect could be achieved with nanoparticles compared to free drug. The nanoparticles were biocompatible and stayed in the tumor for a longer time than a free medicine did, before being eliminated by renal excretion. Overall, the results showed that mesoporous silica nanoparticles are biocompatible, biodegradable drug carriers and that cell specificity can be achieved both in vitro and in vivo.

Delivery of European Cross-Border Health Care and the Relevance and Effects of EU Regulations and Judicial Processes with Reference to Delivery of Drugs and Blood Donor Information Material.

Valtion rajat ylittävät terveyspalvelut Euroopan unionissa sekä Euroopan unionin säädösten merkitys ja vaikutus erityisesti lääkejakeluun ja verenluovuttajille jaettavaan tiedotusaineistoon Valtion rajat ylittävä terveydenhuolto on suuren kiinnostuksen kohteena Euroopan unionissa. Resurssien hyödyntäminen parhaalla mahdollisella tavalla ja tiedon keskittäminen ovat tarpeen terveydenhuollon kustannusten alati noustessa. Terveydenhuoltopalvelut kuuluvat Euroopan sisämarkkinoiden vapaan liikkuvuuden piiriin. Euroopan unionilla ei ole kuitenkaan toimivaltaa säädellä terveydenhuoltojärjestelmiä, vaan sen mahdollisuudet ovat enimmäkseen kansanterveyden edistämisessä ja suojelussa, myös muilla toimialueilla kuin terveydenhuollossa. Tutkimuksen tavoitteena oli tutkia Euroopan unionin säädösten vaikutusta terveydenhuoltosektoriin, erityisesti valtion rajat ylittäviin terveydenhuoltopalveluihin. Erityiskohteena olivat lääkemääräyksen toimittaminen toisen Euroopan unionin jäsenmaan apteekista, resepti-lääkkeiden maahantuonti omaan henkilökohtaiseen käyttöön, sähköisen lääkemääräyksen käyttö kansallisesti ja mahdollisuudet sen käyttöön eri jäsenmaiden välillä, online-apteekkien soveltuvuus Euroopan unionin sisämarkkinoille sekä verenluovuttajille jaettavan tiedotusaineiston yhtenäistämistarve Euroopan unionin alueella. Tutkimuksen osa-alueiden aineisto koottiin vuosina 1999–2003, jolloin Euroopan unioniin kuului 15 jäsenmaata. Apteekit toimittivat useimmiten myös ei-kansalliset, toisessa Euroopan unionin jäsenmaassa annetut lääkemääräykset. Kaikki jäsenmaat rajoittivat lääkemääräyksen vaativien lääkkeiden maahantuontia. Rajoituksia oli maahantuontimäärissä ja -tavoissa. Lisäksi sairasvakuutuskorvausten saaminen ulkomailla lunastetuista reseptilääkkeistä oli hankalaa. Sähköiset lääkemääräykset olivat käytössä vain kahdessa maassa, mutta useissa maissa suunniteltiin niiden kokeilua. Standardit ja käyttöjärjestelmät olivat erilaisia eri maissa. Euroopan unionin alueelle on perustettu online-apteekkeja, joiden toiminta on kuitenkin vaatimatonta. Verenluovuttajille annettava tiedotusaineisto ei missään maassa täyttänyt veridirektiivin vaatimuksia. Tutkimuksen tulokset osoittivat kansallisten käytäntöjen eroavaisuuksien rajoittavan valtion rajat ylittäviä terveydenhuoltopalveluita. Vaikka Euroopan unionin tavoitteena ei ole yhtenäistää terveydenhuoltojärjestelmiä, on tarpeen arvioida uudelleen unionin ja jäsenmaiden välistä työnjakoa. Kansalliset terveydenhuoltojärjestelmät eivät ole erillään Euroopan sisämarkkinoista, jotka merkittävästi vaikuttavat terveydenhuoltoon.

Performance Evaluation of Imaging Systems

Imaging systems have developed latest years and developing is still continuing following years. Manufacturers of imaging systems give promises for the quality of the performance of imaging systems to advertise their products. Promises for the quality of the performance are often so good that they will not be tested in normal usage. The main target in this research is to evaluate the quality of the performance of two imaging systems: Scanner and CCD color camera. Optical measurement procedures were planned to evaluate the quality of imaging performances. Other target in this research is to evaluate calibration programs for the camera and the scanner. Measuring targets had to choose to evaluate the quality of imaging performances. Manufacturers have given definitions for targets. The third task in this research is to evaluate and consider how good measuring targets are.

Predictors and Correlates of Substance Use among Young Men. The Longitudinal “From a Boy to a Man” Birth Cohort Study.

Substance use is one of our most important public health problems. Studying risk factors in a longitudinal study setting helps to identify subgroups of young people at greater risk for substance-use-related problems, and to facilitate targeted prevention efforts. The aim of this thesis was to study childhood predictors and correlates of substance-use-related outcomes among young men in a longitudinal, nationwide birth cohort study. The study population included 10% of all Finnish-speaking boys born in Finland in 1981 (n=2946, 97% of the target population). In 1989, at age eight, valid measures of psychiatric symptoms (Rutter questionnaires and Children’s Depression Inventory) were obtained from parents, teachers and the boys themselves. In 1999, at age 18, boys were reached at their obligatory military call-up (n=2348, 80% of the boys attending the study in 1989). Self-reports of substance use, psychopathology, adaptive functioning (Young Adult Self-Report), and mental health service use were obtained through questionnaires. Information about psychiatric diagnoses from the Military Register (age 18-23 years) and information about offending from the National Police Register (age 16-20 years) were collected in early adulthood (92% of the 1989sample). Boys with childhood conduct, hyperactive, and comorbid conduct-emotional problems had elevated rates of substance use and substance-use-related crime in early adulthood. Depressive symptoms predicted daily smoking, especially among boys of low-educated fathers. Emotional problems predicted lower occurrence of drunkenness-related alcohol use and smoking. Teacher reports on boys’ problem behaviour had the best predictive power for later substance use. At age 18, frequent drunkenness associated with delinquency, smoking and illicit drug use, and having friends. Occasional drunkenness associated with better psychosocial functioning in general compared to boys with frequent drunkenness or without drunkenness-related alcohol use. Illicit drug use without drug offending was not predicted by childhood psychiatric symptoms, but 22% of boys with illicit drug use had a psychiatric diagnosis in early adulthood. Drug offenders, in turn, had psychiatric problems both in childhood and in adulthood. Psychiatric disorders were common among young men with substance-use-related crime. Recidivist crime associated strongly with having a substance use disorder diagnosis according to the Military Register. At age 18, frequent drunkenness was common among boys entering mental health services, but entering substance use treatment was non-existent. According to the findings of this thesis, substance-use-related outcomes accumulate in boys having psychiatric problems both in childhood and in early adulthood. Targeted early interventions in school health care systems, particularly for boys with childhood hyperactive, conduct, and comorbid conduct-emotional problems are recommended. Psychiatric problems and risky behaviours, such as delinquency should always be assessed alongside substance use. Specialized and multidisciplinary care are required for young men who have multiple or complex needs, for instance, for young men with drug offending and recidivist crime. Integrating a substance use treatment perspective with other services where young men are encountered is emphasized.

A Co-Processor Approach for Efficient Java Execution in Embedded Systems

This thesis deals with a hardware accelerated Java virtual machine, named REALJava. The REALJava virtual machine is targeted for resource constrained embedded systems. The goal is to attain increased computational performance with reduced power consumption. While these objectives are often seen as trade-offs, in this context both of them can be attained simultaneously by using dedicated hardware. The target level of the computational performance of the REALJava virtual machine is initially set to be as fast as the currently available full custom ASIC Java processors. As a secondary goal all of the components of the virtual machine are designed so that the resulting system can be scaled to support multiple co-processor cores. The virtual machine is designed using the hardware/software co-design paradigm. The partitioning between the two domains is flexible, allowing customizations to the resulting system, for instance the floating point support can be omitted from the hardware in order to decrease the size of the co-processor core. The communication between the hardware and the software domains is encapsulated into modules. This allows the REALJava virtual machine to be easily integrated into any system, simply by redesigning the communication modules. Besides the virtual machine and the related co-processor architecture, several performance enhancing techniques are presented. These include techniques related to instruction folding, stack handling, method invocation, constant loading and control in time domain. The REALJava virtual machine is prototyped using three different FPGA platforms. The original pipeline structure is modified to suit the FPGA environment. The performance of the resulting Java virtual machine is evaluated against existing Java solutions in the embedded systems field. The results show that the goals are attained, both in terms of computational performance and power consumption. Especially the computational performance is evaluated thoroughly, and the results show that the REALJava is more than twice as fast as the fastest full custom ASIC Java processor. In addition to standard Java virtual machine benchmarks, several new Java applications are designed to both verify the results and broaden the spectrum of the tests.

Physical Realization of an Optical Automatic Target Recognition System

In the modern warfare there is an active development of a new trend connected with a robotic warfare. One of the critical elements of robotics warfare systems is an automatic target recognition system, allowing to recognize objects, based on the data received from sensors. This work considers aspects of optical realization of such a system by means of NIR target scanning at fixed wavelengths. An algorithm was designed, an experimental setup was built and samples of various modern gear and apparel materials were tested. For pattern testing the samples of actively arm engaged armies camouflages were chosen. Tests were performed both in clear atmosphere and in the artificial extremely humid and hot atmosphere to simulate field conditions.